Institute of Reproductive Health/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Wuhan Tongji Reproductive Hospital, Wuhan, 430013, China.
Stem Cell Res Ther. 2021 Oct 29;12(1):553. doi: 10.1186/s13287-021-02621-1.
Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction.
We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.
The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway.
VPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.
研究人类生殖细胞发育和男性不育症严重依赖于小鼠模型。体外分化人类多能干细胞为精原干细胞样细胞（SSCLCs）可作为研究人类生殖细胞和不育症的模型。本研究旨在开发 SSCLC 诱导方案，并评估该方案对 SSCLC 诱导的影响。
我们研究了丙戊酸（VPA）、维生素 C（VC）以及 VPA 和 VC 的组合对 SSCLC 诱导效率的影响，并确定了分化细胞中精原细胞基因的表达。还检测了单倍体细胞和表达减数分裂基因的细胞。进行 RNA-seq 分析比较细胞在分化 0 天和 12 天的转录组，并用 RT-qPCR 确认差异表达基因。我们进一步评估了分化 12 天时组蛋白标记（H3K9ac 和 H3K27me3）的变化。此外，还使用不同的诱导方案评估了来自两名非梗阻性无精子症（NOA）患者的 hiPSC 系的 SSCLC 诱导效率。
在诱导培养基中使用低浓度 VPA 和 VC 的组合在 12 天的分化时最有效地诱导表达几种精原细胞基因的 SSCLCs。高浓度的 VPA 更有效地诱导表达减数分裂基因和单倍体细胞的细胞。RNA-seq 分析表明，SSCLC 的诱导涉及 Wnt 信号通路中上调的基因，并且分化 12 天时的细胞显示出 H3K9ac 的增加和 H3K27me3 的减少。此外，两名来自 NOA 患者的 hiPSC 系显示出低的 SSCLC 诱导效率和 Wnt 信号通路基因表达的减少。
VPA 强有力地促进了人类多能干细胞向 SSCLCs 的分化，涉及 Wnt 信号通路中上调的基因和表观遗传变化。来自 NOA 患者的 hiPSC 系显示出 SSCLC 诱导效率降低和 Wnt 信号通路基因表达减少，提示无精子症睾丸中 SSC 的耗竭可能与 Wnt 信号通路的失活有关。我们开发的 SSCLC 诱导方案为研究人类生殖细胞发育和男性不育症提供了可靠的工具和模型。
